The long term goal of this project is to advance the understanding of protein trafficking and exocytosis. Trafficking of proteins targeted for the apical or basolateral cell surface or for secretion is critical to the function of polariz-ed epithelia. The information that targets a protein to a specific cell surface domain may be an intrinsic property of the protein itself or may involve accessory proteins. Recent observations suggest crosstalk between the vesicle traf-ficking machinery and members of the KCNQ1 K+ charmel complex that consists at least of KCNQ1, KCNE1 and AKAP. Cross talk may occur at the level of common machinery for targeting (SNAREs, KCNEs, AKAPs) and/or at the common signaling pathways (G-proteins, cAMP, Ca2+). Under Aim 1, we will develop a model structure of the KCNQ1 channel complex in proximal tubule and pancreatic acinar cells, determine the solution structure of KCNE1 and model the KCNQ1 complex using chemical crosslinking, other proteomics approaches, NMR analysis and modeling. Under Aim 2, we will determine the basis of tissue-specific trafficking of KCNQ1. We will deter-mine in pancreatic acinar cells, proximal tubules and small intestine which KCNE isoforms are expressed, whether heteromeric assemblies of KCNE subunits occur, where the KCNQ1 complex is located, and whether other acces-sory proteins play a role in targeting. Studies will entail expression and functional proteomics analyses as well as corffocal microscopy. Under Aim 3, we will establish and evaluate 3D and 2D cultures of pancreatic acinar cells. Studies will compare the morphology, gene expression and secretory function through confocal microscopy, RT-PCR, gene array, proteomics and functional analyses including assays for cAMP production, amylase secretion, and for agonist-induced Ca 2+ changes (confocal fluo4 microfluorometry). Under Aim 4, we will determine whether G-protein regulation of exocytosis is downstream of the Ca 2+- and cAMP-dependent steps, identify specific hetero-trimeric G-proteins and their targets that mediate CCK and somatostatin action in acinar cells and elucidate whe-ther SNARE protein(s) interact with activated G protein subunits. Studies will entail the use of antisense nucleo-tides and antibodies to inhibit expression and activity, functional proteomics and in vitro binding assays using GST-tagged SNARE proteins. Completion of these studies will advance our understanding of the mechanisms that affect the targeting of proteins that are destined to be secreted or expressed at specific epithelial surface domains.